One of the more significant scientific and technological advances for the past half-century has been the development of antimicrobial drugs, such as antibiotics and antiviral agents. The widespread availability of these drugs has saved millions of lives and has benefitted mankind in innumerable ways. The only limitation to the usefulness of such drugs has been the evolutionary development of drug-resistant pathogens.
Bacterial pathogens may become resistant to antibiotic drugs in a variety of ways, such as by mutating the target of the drug, by limiting uptake of the drug, or by destroying the drug. Often, the drug target is a protein necessary for the survival and/or proliferation of the pathogen, and resistance to the drug is conferred by means of one or more resistance-conferring mutations in the nucleic acid sequence which encodes the drug target, the resistance-conferring mutations resulting in mutant forms of the drug target in which the drug target loses its affinity for the drug targeted thereagainst while retaining its functionality.
The problem of widespread and ever-increasing bacterial resistance to antibiotics, which now poses a significant threat to public health, has recently been addressed by Harold C. Neu in "The Crisis in Antibiotic Resistance," Science, Vol. 257, pp. 1064-1073 (Aug. 21, 1992). As relayed by Neu, the extensive use of antibiotics over the past several decades has resulted in a proliferation of drug-resistant bacteria. As one example, Neu notes that, in 1941, virtually all strains of Staphylococcus aureus worldwide were susceptible to penicillin G whereas, today, in excess of 95% of S. aureus worldwide are resistant to penicillin, ampicillin, and the antipseudomonas penicillins. As another example, Neu notes that, in 1941, a therapy consisting of 10,000 units of penicillin administered four times a day for 4 days was sufficient to cure patients afflicted with pneumococcal pneumonia whereas, today, a patient could receive 24 million units of penicillin a day and still die of pneumococcal meningitis caused by Streptococcus pneumoniae.
Part of the problem of bacterial resistance to antibiotics stems from the manner in which such drugs have traditionally been developed and used. Typically, a first antibiotic is developed against a substantially uniform, static target (e.g., a single or a small number of pathogenic bacterial strains, a homogenous enzyme preparation, a uniform receptor preparation, or the like) and is then used against an ever-evolving, increasingly heterogeneous target until widespread resistance to the drug develops. Then, a second antibiotic is similarly developed against a resistant, yet similarly uniform and static, form of the target and is substituted for the first antibiotic until, in turn, widespread resistance to it develops. This sequence is usually perpetuated, as new drugs become available, over a period of years as evermore robust, heartier pathogens emerge in response to increasing selective pressure. Even though it has been appreciated that, in many instances, resistance to some drugs will develop over time, the consensus has been that new drugs will become available in the future to successfully combat resistant strains. Unfortunately, this has not always been the case, and the rate at which effective new antibiotics are currently being developed is slower than in the past.
Bacteria are not the only pathogenic microorganisms that have presented a problem to the medical community due to their ability to acquire resistance to drugs targeted thereagainst. Viruses, most notably the HIV virus, have presented a similar problem with respect to antiviral agents. See, e.g., H. Mohri et al., "Quantitation of zidovudine-resistant human immunodeficiency virus type 1 in blood of treated and untreated patients," Proc. Natl. Acad. Sci., U.S.A., Vol. 90, pp. 25-29 (1993); M. Tisdale et al., "Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase," Proc. Natl. Acad. Sci., U.S.A., Vol. 90, pp. 5653-5656 (1993); and R. Yarchoan et al., "Challenges in the therapy of HIV infection," Clinical Perspectives, Vol. 14, pp. 196-202 (1993).
Margaret I. Johnston and Daniel F. Hoth, in "Present Status and Future Prospects for HIV Therapies," Science, Vol. 260, pages 1286-1293 (May 28, 1993), review some of the efforts of researchers to develop anti-HIV agents and report some of the well-accepted explanations as to why such agents have not been fully effective. One such explanation for drug failure is the emergence of drug resistance. Johnston and Hoth note that HIV resistance has been observed for each of the widely used antiretroviral nucleosides used to treat HIV. As an example, Johnston and Hoth refer to one such antiretroviral nucleoside, 3'-azidothymidine (AZT), which was identified in 1984 as being active against HIV in cell culture but which, today, has been observed to lead to resistance in individuals as quickly as 6 months after treatment has begun.
Another example of HIV drug resistance has recently emerged in connection with a new HIV protease inhibitor developed by Merck & Co. See M. Waldholz, "Merck faces dismay over test results: HIV resists promising new AIDS drug," Wall Street Journal (Feb. 25, 1994). No resistance to this drug, which Merck identifies under the trade designation L-735,524, had been observed in cell culture studies prior to human trials; however, during clinical evaluations, indications of resistance emerged.
Viral resistance to antiviral agents is typically conferred by one or more resistance-conferring mutations in the viral nucleic acid sequence encoding the targeted viral protein. Particularly in the case of certain retroviruses, such as the HIV virus, the mutational frequency can be quite high. In fact, in certain individuals infected with the HIV virus, as much as 20% of the viruses are found to contain mutations. See Wain-Hobson, "The fastest genome evolution ever described: HIV variation in situ," Current Opinion in Genetics and Development, 3:878-883 (1993). This high mutational frequency is primarily attributable to the operation of the HIV reverse transcriptase enzyme, which is used to convert single stranded viral RNA into double stranded DNA as part of the viral life cycle but which lacks an editing mechanism. Because of its high mutational frequency, the HIV virus has been characterized as "a perpetual mutation machine," id. at 881. In fact, there is a widespread belief in the art that, at least with respect to the HIV virus and similar viruses, a virtually unlimited number of distinct evolutionary escape pathways exist for any protein with respect to practically any drug. See e.g., Honess et al., "Single Mutations at Many Sites within the DNA Polymerase Locus of Herpes Simplex Viruses Can Confer Hypersensitivity to Aphidicolin and Resistance to Phosphonoacetic Acid," J. gen. Virol., Vol. 65, pp. 1-17 (1984); Saag et al., "Extensive variation of human immunodeficiency virus type-1 in vivo," Nature, Vol. 334, pp. 440-444 (Aug. 4, 1988); Richman, "HIV Drug Resistance," Annu. Rev. Pharmacol. Toxicol., Vol. 32, pp. 149-164 (1993); and Wain-Hobson, "The fastest genome evolution ever described: HIV variation in situ," Current Opinion in Genetics and Development, 3:878-883 (1993). Alternatively stated, there appears to be no recognition in the art that, at least with respect to certain drugs, the number of different resistance-conferring mutations available to a given protein may be quite limited. Consequently, HIV drug resistance (and, more broadly stated, viral drug resistance) is presently considered by the art to be an intractable problem.
One way in which prospective drugs have traditionally been evaluated prior to clinical use is by a technique commonly referred to as cell-culture selection. To test antiviral agents using cell-culture selection, one typically grows a targeted virus on a host cell line in the presence of a prospective drug. Progeny viruses are then serially passaged in the host cell line in the presence of an increasing concentration of the prospective drug to select drug-resistant strains. An exemplary application of cell-culture selection to prospective drug evaluation is disclosed in Tisdale et al., "Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase," Proc. Natl. Acad. Sci., U.S.A., Vol. 90, pp. 5653-5656 (June 1993). In Tisdale, MT-4 cells were infected with either wild-type HIV-1 or an AZT-resistant strain derived from wild-type HIV-1 and exposed to low concentrations of (-)-2'-deoxy-5-fluoro-3'-thiacytidine (FTC). Progeny virus was recovered and serially passaged in MT-4 cells in the presence of increasing FTC concentration. By the fourth passage of the wild-type progeny and only the second passage of the AZT-resistant progeny, IC.sub.50 (50% inhibitory concentration) values exceeded 50 .mu.M. When tested at higher compound concentrations, the IC.sub.50 values of passage 6 virus were in excess of 250 .mu.M. Based on the rapid emergence of resistant virus, Tisdale et al. postulated that the therapeutic value of FTC, except possibly in combination with other HIV-1 inhibitors, may be limited.
Another exemplary application of cell-culture selection to prospective drug evaluation is disclosed in Taddie et al., "Genetic Characterization of the Vaccinia Virus DNA Polymerase: Identification of Point Mutations Conferring Altered Drug Sensitivities and Reduced Fidelity," Journal of Virology, Vol. 65, No. 2, pp. 869-879 (February 1991). In Taddie, wild-type vaccinia virus was chemically mutagenized with nitrosoguanidine and then serially passaged through African green monkey BSC40 cells in the presence of 85 .mu.M aphidicolin in an effort to isolate aphidicolin-resistant virus.
A technique analogous to the cell-culture selection technique described above for antiviral agents has been used to test the efficacy of antibiotics. See e.g., Handwerger et al., "Alterations in Penicillin-Binding Proteins of Clinical and Laboratory Isolates of Pathogenic Streptococcus pneumoniae with Low Levels of Penicillin Resistance," The Journal of Infectious Diseases, Vol. 153, No. 1, pp. 83-89 (January 1986) (wherein clones resistant to benzylpenicillin were selected by serial passage on blood agar plates in two-fold increasing concentrations of benzylpenicillin).
In testing both antibiotics and antiviral agents in the above manner, most investigators have focused primarily on the speed with which marked resistance to the prospective drug emerges and on the IC.sub.50 values of the prospective drug as the key factors used to gauge the potential therapeutic value of the drug. Typically, the more rapid the development of resistance, the less desirable the prospective drug has been adjudged. Thus, in evaluating prospective drugs, the art focuses primarily on the rate of mutation, without regard to the nature or number of different drug-resistant mutants.
Although widely used, cell-culture selection is fraught with limitations. One such limitation is that the cell-culture technique itself may be unfairly biased against the selection of certain mutant strains that would have emerged in vivo. See Meyerhans et al., "Temporal Fluctuations in HIV Quasispecies In Vivo Are Not Reflected by Sequential HIV Isolations," Cell, Vol. 58, pp. 901-910 (Sep. 8, 1989). In the aforementioned Meyerhans article, HIV-1 isolates obtained from a patient over a two and one-half year period as well as from cultured peripheral blood mononuclear cells (PBMC) were analyzed and compared. The tat gene from the respective isolates was amplified by polymerase chain reaction (PCR), and amplified DNA was cloned into a mammalian expression vector. Twenty clones from each sample were sequenced. The HIV quasispecies--populations of viral genomes--showed significant differences between corresponding in vivo and in vitro samples. For example, the major form of one in vivo isolate was derived from the minor form of a corresponding in vitro isolate. From these results, Meyerhans et al. were led to conclude that "to culture is to disturb."
Another limitation inherent in cell-culture selection is that one is not assured that each and every mutation that may emerge in vivo will be generated for possible selection. Still another limitation inherent in cell-culture selection is that certain drug-conferring mutations may be masked by the simultaneous occurrence of lethal mutations in genes other than the gene under observation. This is because cell-culture selection affords no means for restricting mutagenesis to the gene under observation.
In UK Patent Application No. 2,276,621, published Oct. 5, 1994, and incorporated herein by reference, there is described a chromogenic assay said to be useful in the identification and isolation of drug-resistant HIV protease mutants. The assay is also said to be useful in the screening of new inhibitors of HIV protease, e.g., inhibitors not affected by drug-resistance of the HIV protease. The subject color screening assay contains a vector comprising a regulatable promoter which controls the transcription of two adjacent structural sequences, one sequence coding for HIV protease or a mutant thereof, the other sequence coding for beta-galactosidase with an amino acid substrate insert cleavable by HIV protease.
Unfortunately, as far as the present inventors are aware, the aforementioned chromogenic assay has had limited success in identifying, in vitro, drug-resistant strains that were later isolated following clinical use. The present inventors believe that the poor predictive nature of the aforementioned chromogenic assay is due, to a considerable extent, to the lack of authenticity in the HIV-protease/beta-galactosidase construct used therein. In other words, in the aformentioned chromogenic assay, the protease mutant need only cleave the protease/beta-galactosidase fusion protein at a single, artificial, cleavage site within the beta-galactosidase protein for a positive result to be registered in the assay; in contrast, in the native HIV polyprotein, the protease must cleave the polyprotein at a number of sites, e.g., at least three sites to activate HIV reverse transcriptase. The present inventors believe that these variations in the authenticity of the nature and number of cleavage sites in the construct of the above-described chromogenic assay effectively render the assay unreliable.
Consequently, for at least the above reasons, there are a number of reported instances in which drug-resistant strains have been observed in vivo which were not predicted by cell-culture studies. See e.g., Smith et al., "Resumption of Virus Production after Human Immunodeficiency Virus Infection of T Lymphocytes in the Presence of Azidothymidine," Journal of Virology, Vol. 61, No. 12, pp. 3769-3773 (December 1987) (reporting that no AZT resistance in the HIV virus was observed following cell-culture selection); Larder et al., "Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity," Proc. Natl. Acad. Sci., U.S.A., Vol. 86, pp. 4803-4807 (July 1989) (reporting that no AZT resistance in the HIV virus was observed following cell-culture selection but noting the presence of AZT-resistant isolates following clinical use); and Larder et al., "Zidovudine-Resistant Human Immunodeficiency Virus Selected by Passage in Cell Culture," Journal of Virology, Vol. 65, No. 10, pp. 5232-5236 (October 1991) (noting that attempts to select zidovudine-resistant strains of HIV in cell culture using wild-type HIV have been unsuccessful and reporting that zidovudine-resistant strains similar to those found clinically were obtained by cell-culture selection of HIV variants constructed by site-directed mutagenesis).
Other limitations with cell-culture selection are that (1) stringent handling conditions must be used to avoid safety problems, since intact pathogens are required to be used; and (2) the cell-culture technique itself is very time consuming (and, hence, expensive) since several passages are usually required, each passage typically taking a number of days.
As alluded to above, because drug resistance is so common, many researchers have assumed that, in virtually every instance in which drug resistance occurs, there are a great many parallel evolutionary escape pathways by which drug resistance is or may be conferred. See Saag et al., "Extensive variation of human immunodeficiency virus type-1 in vivo," Nature, Vol. 334, pp. 440-444 (Aug. 4, 1988) (reporting that, following the sequential isolation of HIV virus from two chronically infected individuals, a remarkably large number of related but distinguishable genotypic variants had evolved in parallel); and Honess et al., "Single Mutations at Many Sites within the DNA Polymerase Locus of Herpes Simplex Viruses Can Confer Hypersensitivity to Aphidicolin and Resistance to Phosphonoacetic Acid," J. gen. Virol., Vol. 65, pp. 1-17 (1984) (reporting that hypersensitivity of Herpes Simplex virus to aphidicolin is a common consequence of single, well-separated mutations).
In fact, the problem of drug resistance has grown to such a level that, with respect to pathogens like HIV, some researchers have concluded that future prospects for efficient therapy and prevention are bleak. See Wain-Hobson, "The fastest genome evolution ever described: HIV variation in situ," Current Opinion in Genetics and Development, Vol. 3, pp. 878-883 (1993) (explaining that the high genetic variability of the HIV virus and the high viral load of the HIV virus raise questions as to whether there are any limits to HIV variation).
Notwithstanding these pessimistic forecasts, new drugs and therapies are continuing to be explored. However, the identification of potential new drugs continues to involve evaluating possible therapeutic agents against a single, static, pathogenic target. Techniques increasingly being used to identify such potential new drugs include rational drug design and combinatorial screening. In rational drug design, the conformational and chemical structure of a desired binding site on a target compound is identified, and prospective drugs are designed and/or evaluated based on their ability to function as a binding partner for the binding site on the single target compound. Exemplary applications of rational drug design are discussed in the following patents and publications, all of which are incorporated herein by reference: U.S. Pat. No. 5,300,425; U.S. Pat. No. 5,223,408; and Roberts et al., "Rational Design of Peptide-Based HIV Proteinase Inhibitors," Science, Vol. 248, pp. 358-361 (Apr. 20, 1990).
In combinatorial screening, various combinatorial arrangements of short oligonucleotide sequences, amino acid sequences, or other organic compounds are screened as prospective binding partners for a binding site on a single target compound. Exemplary applications of combinatorial screening are discussed in the following patents and publications, all of which are incorporated herein by reference: U.S. Pat. No. 5,288,514; U.S. Pat. No. 5,258,289; Barbas, III et al., "Semisynthetic combinatorial antibody libraries: A chemical solution to the diversity problem," Proc. Natl. Acad. Sci., USA, Vol. 89, pp. 4457-4461 (May 1992); and Alper, "Drug Discovery on the Assembly Line," Science, Vol. 264, pp. 1399-1401 (Jun. 3, 1994).
Recently, the idea of co-administering two or more drugs directed at different proteins of a given pathogen, specifically HIV, ("combination therapy") has emerged as a possible way of overcoming the problem of drug resistance. Examples of approaches utilizing two or more drugs targeted against different proteins of a single pathogen are discussed in Kageyama et al., "In Vitro Inhibition of Human Immunodeficiency Virus (HIV) Type 1 Replication by C.sub.2 Symmetry-Based HIV Protease Inhibitors as Single Agents or in Combinations," Antimicrobial Agents and Chemotherapy, Vol. 36, No. 5, pp. 926-933 (May 1992) and in "Pharmaceutical Consortium to Begin Clinical Trials of Combined AIDS Drugs," Wall Street Journal (Apr. 14, 1994). In the Kageyama article, for example, the effect of combinations of certain C.sub.2 symmetry-based HIV protease inhibitors, such as A75925, A77003 and A76928, with AZT or ddI (reverse transcriptase inhibitors) was investigated in vitro. For certain combinations of drugs, encouraging in vitro results were observed. (For example, A75925 combined with AZT resulted in virtually complete suppression in vitro).
The present inventors believe, however, that combination therapy of the type described above will ultimately fail in vivo due to the emergence, under selective pressure, of pathogens containing resistant forms of all targeted proteins. The emergence of such pathogens may even be hastened in the event that genomes with resistance-conferring mutations in different targeted proteins recombine with one another to form multiply resistant pathogens.
Another approach that has recently emerged as a possible way of overcoming the problem of drug resistance is to co-administer two or more drugs directed at different active sites on the same protein of a given pathogen ("convergent combination therapy"). An example of this approach is disclosed in Chow et al., "Use of evolutionary limitations of HIV-1 multidrug resistance to optimize therapy," Nature, Vol. 361, pp. 650-654 (Feb. 18, 1993). In the Chow article, mutations in different active sites on the HIV-1 reverse transcriptase gene conferring multiple drug resistance to wild-type inhibitors of reverse transcriptase were constructed to determine whether multiple drug resistance is incompatible with viral replication. Viruses containing combinations of mutations conferring resistance to AZT, ddI and a pyridinone were reported to be incapable of viral replication. Chow et al. postulated that the existence of these mutant viruses indicated that evolutionary limits exist to restrict the development of multiple drug resistance. However, it was later pointed out in Chow et al., "HIV-1 error revealed," Nature, Vol. 364, page 679 (Aug. 19, 1993) that the multiply-drug-resistant mutant referred to above had unintended mutations which were responsible for its lack of viability. It was further pointed out in Emini et al., "HIV and multidrug resistance," Nature, Vol. 364, page 679 (Aug. 19, 1993) that the multiply-drug-resistant Chow mutant exhibited growth kinetics in the presence of inhibitors similar to wild-type virus while still exhibiting a multiply resistant phenotype.
The present inventors believe that convergent combination therapy of the type described above is flawed because each and every drug used therein is targeted against different sites on the same static species of the protein, namely the original or wild-type species. In other words, none of the drugs of the aforementioned convergent combination therapy are specifically directed against mutant, drug-resistant forms of the protein that may emerge under selective pressure, nor are any of the drugs of the aforementioned convergent combination therapy specifically directed against mutations which confer resistance to any of the other drugs of the combination. As a result, there can be no assurance that every mutant form of the protein that is resistant to one of the drugs of the combination will be rendered inactive by any of the other drugs of the combination.
Thus, as can be seen, the techniques utilized in the prior art to screen and compare prospective drugs, as well as to design clinical therapies, have been either ineffectual or impractical.
Accordingly, there presently exists a need for effective therapies against pathogenic microorganisms to overcome the problem of drug resistance. In addition, there is a need to predict, prior to clinical administration of a prospective drug, all possible, first-generation, drug-resistant, biologically-active mutants which could emerge in response to the drug, to compare drugs in terms of the ease with which resistance develops against them, and to identify drugs effective against such drug-resistant mutants. Further, there is a need for an in vitro technique that can be used to predict drug-resistant, biologically-active mutants of a protein to a subject drug in a manner that it is more time-efficient and economical than conventional cell-culture selection techniques.